Shanukha Thivyanath Thursday November. 14/ 2013 Summary of Topics Covered in Class:- Structure of DNA (Chemical Composition)- Edwin Chargaff's Data (Notion of Complementary Nitrogenous Bases) - Wilkins and Franklin (Double Helix was suggested through x-ray crystallography results, no link to nitrogenous bases, therefore analysis was incomplete) * Personal Notes had to be taken from pages 276-277 - Watson and Crick (Accepted theory of Double Helix) Notes:Structure of DNA- DNA structure: the monomer of DNA is the nucleotide- The phosphate and deoxyribose form the rails of the molecule; while these can be either a purine or pyrimidine nitrogenous base.(Prime 3 and 5 refers to carbons in deoxyribose) Edwin Chargaff's Data (Early 1950s)- Did not believe there were equal amounts of the 4 nitrogenous bases like everyone else.- He looked at purine vs. pyrimidine (nitrogenous base) composition in different samples of DNA- He measured molar ratios of the various nucleotides- Ratios of A:T and G:C were very similar in many species investigated- Thus suggesting the pairing of these bases with each other for some reason- This lead to the notion of complementary pairs!A approx= T--> in % of base in DNAG approx = C--> in % of base in DNA Wilkins and Franklin: Another Piece of the Puzzle- New technology of their time: X-ray crystallographyX ray crystallography: X rays hitting a sample of a compound (solid crystal form); Atoms in crystal deflect the x-rays in a certain way making a pattern on a photographic plate. The pattern is analyzed to determine molecular structure of the original.- W & F used crystallography to study the shape of the DNA molecule.- Both worked independently, but came to similar conclusions.- W--> produced some preliminary crystallographs of DNA that suggested its helical structure- F--> prepared purer crystallized DNA samples and was able to produce clear crystallographs. Pattern was in the shape of an X.--> Suggested the sugar phosphate backbones of DNA faced the outside of the molecule- DNA had the diameter of 2nm and 1 turn of the helix was 3.4 nm in length- She couldn't explain how the nitrogenous bases were in the centre of the helix Structure of DNA- Watson andCrick- Drawing on their own research and that of others they suggested a structure for the DNA molecule- Consisting of a double helix- Purine, pyrimidine pairing (AT, GC) from one helix to another- Pairing was held together by two hydrogen bonds b/w A and T and three bonds b/w G and C- Helices were arranged anti- parallel to one another (one spiraled up to the right, the other down to the right)
HOMEWORK: Notes on Wilkins and Franklin on pg 276-277
(My notes are written above: you're welcome!)
Martha G.
Monday November 18, 2013
DNA Replication
The Discovery of DNA Replication
Meselon and Stahl
Investigated whether DNA replication is conservative (one strand is 100% the old DNA and the second strand is 100% new DNA) or semi-conservative (both strands of DNA are 50% old DNA and 50% new DNA)
They used E. coil and grew it in heavy nitrogen, 15N, medium (for example a petri dish) so all nitrogenous bases of the DNA would be labelled
They then transferred the bacteria to a 14N medium
They compared the density of the bacteria in the 15N and 14N mediums which reflected the semi-conservative process
DNA polymers consist of nucleotides linked by 3’ – 5’ phosphodiester bonds
Chemically, the concentration of purine equals the concentration of pyrimidine in DNA
[A] = [T] and [G] = [C]
Heating DNA slightly changes its physical properties without breaking the 3’ – 5’ phosphodiester bonds
The DNA would uncoil and unzip under heat
DNA molecules have a double helical structure
The DNA double helix is unzipped with the use of enzymes
DNA polymerase adds new nucleotides to the now unzipped portions
New nucleotides are attached to the 3’ end of the newly synthesized strand by a DNA polymerase
Bacteria have 3 types of DNA polymerase while eukaryotes have 5 types
The steps of DNA Replication:
DNA gyrase unwinds the DNA from its helical shape
DNA helicase unzips the DNA by breaking the hydrogen bonds that hold it together
Single stranded binding proteins (SSBs) are bound to the single stranded DNA section to keep them from pairing up or re-annealing again
Primase puts down RNA primers that will indicate to DNA polymerase III to start replication
*DNA cannot fully unwind due to its large size so replication occurs immediately when areas of the DNA are exposed as a single strand*
Replication forks and within bacteria, replication bubbles form
5. DNA polymerase III (5’ – 3’) adds the complementary nucleotides to the 3’ end of the new strand (called the leading strand) 6. DNA polymerase I cuts out the RNA primers and replaces them with appropriate DNA nucleotides 7. DNA ligase joins the gaps between the lagging strand by creating phosphodiester bonds 8. DNA polymerase I and III proofread the complementary strands 9. DNA polymerase II fixes errors in the DNA
Nejaha Abdulmalik
November 19th 2013
Today was a study period for the Unit test
Make sure to review everything from the beginning of chapter 3 to chapter 5
Shanukha Thivyanath November 19, 2013
Topics Covered in Class:
- Recall of DNA Replication (10 steps)
- Easy to Understand Diagrams of the process
Process of DNA Replication: Recall:
DNA in prokaryotes= circular, DNA in eukaryotes = linear
1. Hydrogen bonds hold the two strands of DNA together and it is twisted in the helical shape.
2.DNA gyrase unwinds DNA.
3. DNA helicase unzips DNA by breaking those hydrogen bonds.
4. Now you have two single stranded sections BUT they have a natural tendency to re-anneal (pair up again.
5.To keep them seperate, single stranded binding proteins (SSBs) are bound.
6. Enzyme primase puts down RNA primers that will indicate to DNA polymerase III as starting point of replication.
7. DNA polymerase III (5' To 3') adds the complementary nucleotides to the 3' end of the new strand- this is called leading strand vs lagging strand (Okazaki fragments), that are built discontinuously from the replication fork.
8. DNA polymerase I excises (cuts out) the RNA primers and replaces them with appropriate deoxyribonucleotides (DNA nucleotides)
9. DNA ligase joins the gaps between the Okazaki fragments by creating phosphodiester bonds.
10. Finally, DNA polymerase I and III proof read the complementary strands.
Diagrams of the Process (Enlarge the photos in edit to see them clearly!) Homework: Pg. 294 Q- 1, 3, 4, 6, 8 + Pg. 298 Q- 1, 3
Martha G.
November 25, 2013 Protein Synthesis
Beadle and Tatum
Came up with the one gene one enzyme hypothesis which states each gene is unique and codes for the synthesis of a single enzyme
Experiment:
Neurospora (bread mould) can generally be grown on a medium of sugar, inorganic acids and salts, ammonium compounds and biotin
The reasoned that Neurospora used these chemicals to make the complex chemicals needed for growth and reproduction
They used x-rays to cause mutations in many samples of the mould cells then took the mutated cells and saw that some did not grow in the original medium anymore
The radiation produced mutant Neurospora which required additional specific nutrient supplements in order to grow in the medium
Their hypothesis:
A mutation has blocked a metabolic step leading to the synthesis of a specific compound
Process of Protein Synthesis
DNA doesn't replicate, it encodes to be decoded
DNA is synthesized during replication but it's transcribed during protein synthesis:
DNA acts as an original tape, preserving information while temporary transcripts make use of the information
The transcripts are made in the nucleus and consists of RNA molecules called mRNA
The mRNA is then translated into polypeptides in the cytoplasm which eventually becomes active proteins
There are three major classes of RNA:
mRNA - The information that is transcribed into the final protein
tRNA - Transfers appropriate amino acids to the ribosomes determined by mRNA
rRNA - Structural components of the ribosomes site of protein synthesis
STAGE ONE: Transcription
Information is transcribed onto another medium (from DNA to RNA)
RNA polymerase transcribes DNA and read 3' to 5'
The synthesized single strand of RNA is complementary to the one strand of DNA which essentially is mRNA
mRNA contains coding information for a specific protein
Post-transcriptional Changes:
Addition of 5' cap consisting of 7 G's which protects the mRNA from digestion of enzymes
Removal of introns (non-coding regions and useless) using spliceosomes
Addition of a poly-Adenine tail (20 to 30 long) which extends the life of the mRNA molecule
Overall Process of Transcription
RNA polymerase does not require a primer
RNA polymerase begins unzipping the DNA at an initiation sequence called a promoter region (TAC)
RNA polymerase reads 3' to 5' and complements the DNA strand
The first bit of "writing" is the 5' UTR (untranslated region) followed by the exons and introns then ends with 3' UTR which is essentially mRNA
It proceeds until it reaches the termination sequence signal in RNA is released and processed
Transcription -------> making mRNA and post-transcriptional changes
HOMEWORK ---> 7.2 Questions #1 - 6
---> If the diagrams in class is not in your possession, then copy it off of a friend!!!
Annie Vimalanathan
November 26, 2013
7.3 Translation
At the ribosome
We read the mRNA in the 5' to 3' direction
Also read mRNA in TRIPLETS aka CODONS
Begin at start codon [AUG] found at the beginning of an exon
There are 3 stop codes/ codons [UGA,UAA,UAG]
1) What is involved
AMINOACYL SITE a.k.a the A site is for READING the mRNA and retrieving the correct tRNA
PEPTIDYL SITE a.k.a the P site is for HOLDING onto the amino acid and forming peptide bonds
EXIT SITE is a.k.a the E site is where the empty tRNA is moved, then released.
As the ribosome moves along the mRNA, it will read it and each codon on the mRNA will go into each site [ A site --> P site --> E site]
2) Transfer RNA
tRNAs are the smallest types of RNA
On the bottom (opposite of the 3' end) you will find the ANTICODON - a three nucleotide segment that pairs with a codon in an mRNA
tRNAs are active or non active, which is determined by whether it has an amino acid attached or not
This process of adding an amino acid to a tRNA is called aminoacylation
The a.a is added to the 3' end of the tRNA via an ester bond
It is done by an enzyme called synthetase
The final product after is called an aminoacyl-tRNA
Step by Step:
INITIATION:
Small ribosomal subunit binds to mRNA and scans it until it finds the start codon [AUG]
[AUG] is sitting in the P site
Active tRNA enters the P site and matches its ANTICODON to the CODON
This matching signals for the large ribosomal subunit to bind
ELONGATION:
The ribosome will read the mRNA codon by codon. This is also known as the reading formula
A site "reads" the second CODON. The correct aminoacyl - tRNA binds in the A site
Peptidyl transferase breaks the ester bond of the 1st tRNA and creates the peptide bond that connects the 1st amino acid to the 2nd amino acid (that is still connected to the 2nd tRNA)
Ribosome SHIFTS the reading frame
non-active tRNA enters into the E site, then exits the ribosome
2nd tRNA enters into the P site
3rd CODON enters into A site ... (repeat step 5-7) until a stop codon enters
TERMINATION:
Stop codon enters into the A site
Instead of an aminoacyl- tRNA entering, a protein release factor binds to the site
Polypeptide is released from the ribosome. Ribosome subunits separate and protein release factor and mRNA are released
We had a supply in class and used the time to:
-Read page 340-345 and make notes
-Do questions 1-8 on page 345
-Work on our "build" project
Martha G.
December 02, 2013
CONTINUATION OF LESSON ON NOVEMBER 28TH!
Example 2: trp Operon
Found in E.coli
It needs tryptophan to produce proteins
Is a positive control system:
The gene of the enzymes are usually "on"
When tryptophan is present, trp binds to the repressor
This creates a trp - trp repressor complex that binds to the operator region and prevents transcription
This system helps the bacteria survive via limiting energy used to manufacture enzyme only when needed (i.e. when trp is not present)
In this example:
Trp operon has five genes that code for five polypeptides that make three enzymes used to synthesize trp
Repressor protein = inactive trp repressor and tryptophan
Under Normal Conditions:
The cell needs trp.
The trp repressor proteins is inactive
RNA will transcribe
The concentration of trp will increase
When trp is present:
Trp will bind to the inactive repressor forming a trp - trp repressor complex which binds to the operator
RNA polymerase cannot transcribe
The concentration of trp will decrease
HOMEWORK: Build Project !!!! Shanukha Thivyanath December 4, 2013
Topics Covered in Class:
- Notes on Plasmid Construction
- Notes on Plasmid Mapping
- Examples of Plasmid Mapping Questions
Plasmid Construction: How Plasmids are made
- A Plasmid contains numerous sites for restriction enzymes to act
- Are single super-coiled circular pieces of DNA inside bacteria
- Not the genomic DNA of the bacteria a specific characteristic (gives them an advantage) i.e ampicillin
-Plasmids can be designed as vehicles to transfer genetic information into a cell. These are called vectors. i.e used in insulin production
- A cell that can take up foreign DNA and express the products of the gene are called competent.
Plasmid Mapping - A restriction map is a diagram showing the restriction enzyme sites on a plasmid - It includes the distances (measured in base pairs) between the sites
- This allows molecular biologists to determine which plasmids are best suited to create a given recombinant DNA
To create a map. scientists first cut the plasmid with the different restriction enzymes (alone and in combination) and determine the lengths of the fragments formed by cleaving (using gel electrophoresis)
-They then use the fragment length data to create their map vectors are created by using restriction enzymes to insert the gene of interest.
Things to Consider when using Plasmids:
-Plasmids may have many restriction sites, but the one you use must occur only once (and have a sticky end if possible).
- The point of insertion should be downstream of an existing control sequence like lac or trp operons
-This allows for control of eventual gene product
- The gene of interest must have no introns or control sequences
- Genes must insert in the correct direction
Examples with Diagrams
Homework: Finish Plasmid Mapping Worksheet, front and back. **Try Level 3 Questions! Quiz is coming soon!
Saad Sheikh
December 5, 2013
Gel Electrophoresis
Gel electrophoresis is a technique used to separate pieces of DNA based on their size (kb) kilobases
samples are inserted into each well
one sample is the control: it has fragments of known molecular sizes aka "the standard" - aka standard ladder
set up the voltage standard - positive at the bottom, negative at the
Recall: DNA is negatively charged due to the phosphate groups
when the current is turned on, the negatively charged fragments will travel towards the positive electrode
the shorter the fragment, the faster it will travel through the gale (made of agarose)
to see the DNA fragments, we use a loading dye
it is made up of a dye (Ethidium bromide) + glycerol (heavy molecule)
gel electrophoresis can be used for protein separation as well
it would use gels made up of a polyacrylamide (smaller pores)
samples are applied to each of the wells, with an "X" sample containing fragments of known molecular mass (standard)
Apply the voltage across the gel with the positive at the bottom and the negative at the top
gel electrophoresis involves the separation of charged molecules on the basis of size/mass
the medium used is some kind of gel, that acts as filter screen or sieve
DNA is negatively charged due to the phosphates, and may be combined with SDS (a type of detergent)
Once loaded into the gel, a charge will attract the DNA towards the positive end with the smallest molecules moving the most quickly
the gel can be thought of as a forest, and the DNA samples like people running through
if there are more people holding hands together (10 or 20) it will be harder (slower) to run through the woods
the people that are running alone or only in pairs will be able to run faster through the woods
Purpose: to amplify a gene fragment (like one for insulin)
Given a short segment of DNA, heat it to 95 degrees Celsius (replaces gyrase and helicase) until it dissociates into single stranded (ssDNA)
Cool it to about 57 degrees Celsius. Incubate the single stranded DNA with a forward and a reverse DNA primers (5' - 3'). This allows the primers to anneal (stick to) the ssDNA
Heat it to 72 degrees Celsius, then add Taq polymerase (heat insensitive) and allow new complementary strands to form. (Why can't you use DNA polymerase III? Because the temperature is too high)
Heat the strands up to 95 degrees Celsius to dissociate the new pieces and the templates
Cool it to 57 degrees Celsius again and allow the primers to anneal, and heat to 72 degrees Celsius to allow Taq to replicate the DNA (Repeat the heating/cooling, synthesizing)
After the second cycle, the new pieces have gone from primer to primer exactly. They are shorter than either the original DNA of the first copy.
Continued cycling causes an exponential increase in the number of short but "consistent"/ constant length pieces. Eventually the proportion of strands that are "odd"/ variable lengths will be so small as to be insignificant
Applications of PCR:
forensic criminal investigations
DNA in a hair follicle or saliva is VERY LITTLE
Use PCR to amplify the DNA so that various tests can be used on it (ex. paternity tests)
genetic testing
Restriction Fragment Length Polymorphism (RFLP)
a technique in which organisms may be differentiated by analysis at patterns derived from digestion of their DNA
if two organisms differ in the distance between sites of digestion of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme
patterns generated can be used to differentiate species (and even strains) from one another
VNTRs (variable number of tandem repeats) aka microsatelites
a noncoding region of DNA
often used in RFLP
a sequence of base pairs that repeat over and over (eg. TAGTAGTAGTAGTAG)
between individuals, VNTRs can vary in length and position within the genome
DNA Sequencing
a sequence of DNA is obtained and divided into four test tubes
Each tube contains everything necessary for DNA replication
also each tube also has a small percentage of nucleotides made with ddDNA (deoxyribonucleic acid)
Each tube has different ddDNA nucleotide
As the DNA is synthesized, occasionally a ddDNA is used
this stops the synthesis at that point due to the absence of an -OH and Carbon #3 of the deoxyribose
Each tube has various sized pieces of DNA each stopped at the same base
By running each sample in parallel in gel, each piece can be separated in gel
Shanukha Thivyanath
Thursday November. 14/ 2013
Summary of Topics Covered in Class:- Structure of DNA (Chemical Composition)- Edwin Chargaff's Data (Notion of Complementary Nitrogenous Bases)
- Wilkins and Franklin (Double Helix was suggested through x-ray crystallography results, no link to nitrogenous bases, therefore analysis was incomplete) * Personal Notes had to be taken from pages 276-277
- Watson and Crick (Accepted theory of Double Helix)
Notes:Structure of DNA- DNA structure: the monomer of DNA is the nucleotide- The phosphate and deoxyribose form the rails of the molecule; while these can be either a purine or pyrimidine nitrogenous base.(Prime 3 and 5 refers to carbons in deoxyribose)
Edwin Chargaff's Data (Early 1950s)- Did not believe there were equal amounts of the 4 nitrogenous bases like everyone else.- He looked at purine vs. pyrimidine (nitrogenous base) composition in different samples of DNA- He measured molar ratios of the various nucleotides- Ratios of A:T and G:C were very similar in many species investigated- Thus suggesting the pairing of these bases with each other for some reason- This lead to the notion of complementary pairs!A approx= T--> in % of base in DNAG approx = C--> in % of base in DNA
Wilkins and Franklin: Another Piece of the Puzzle- New technology of their time: X-ray crystallographyX ray crystallography: X rays hitting a sample of a compound (solid crystal form); Atoms in crystal deflect the x-rays in a certain way making a pattern on a photographic plate. The pattern is analyzed to determine molecular structure of the original.- W & F used crystallography to study the shape of the DNA molecule.- Both worked independently, but came to similar conclusions.- W--> produced some preliminary crystallographs of DNA that suggested its helical structure- F--> prepared purer crystallized DNA samples and was able to produce clear crystallographs. Pattern was in the shape of an X.--> Suggested the sugar phosphate backbones of DNA faced the outside of the molecule- DNA had the diameter of 2nm and 1 turn of the helix was 3.4 nm in length- She couldn't explain how the nitrogenous bases were in the centre of the helix
Structure of DNA- Watson and Crick- Drawing on their own research and that of others they suggested a structure for the DNA molecule- Consisting of a double helix- Purine, pyrimidine pairing (AT, GC) from one helix to another- Pairing was held together by two hydrogen bonds b/w A and T and three bonds b/w G and C- Helices were arranged anti- parallel to one another (one spiraled up to the right, the other down to the right)
HOMEWORK: Notes on Wilkins and Franklin on pg 276-277
(My notes are written above: you're welcome!)
Martha G.
Monday November 18, 2013
DNA Replication
*DNA cannot fully unwind due to its large size so replication occurs immediately when areas of the DNA are exposed as a single strand*
5. DNA polymerase III (5’ – 3’) adds the complementary nucleotides to the 3’ end of the new strand (called the leading strand)
6. DNA polymerase I cuts out the RNA primers and replaces them with appropriate DNA nucleotides
7. DNA ligase joins the gaps between the lagging strand by creating phosphodiester bonds
8. DNA polymerase I and III proofread the complementary strands
9. DNA polymerase II fixes errors in the DNA
HOMEWORK! à Read 6.4
Resources:
http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swf
http://sites.fas.harvard.edu/~biotext/animations/replication1.swf
http://highered.mcgraw-hill.com/sites/0072943696/student_view0/chapter3/animation__dna_replication__quiz_1_.html
Nejaha Abdulmalik
November 19th 2013
Today was a study period for the Unit test
Make sure to review everything from the beginning of chapter 3 to chapter 5
Shanukha Thivyanath
November 19, 2013
Topics Covered in Class:
- Recall of DNA Replication (10 steps)
- Easy to Understand Diagrams of the process
Process of DNA Replication:
Recall:
DNA in prokaryotes= circular, DNA in eukaryotes = linear
1. Hydrogen bonds hold the two strands of DNA together and it is twisted in the helical shape.
2.DNA gyrase unwinds DNA.
3. DNA helicase unzips DNA by breaking those hydrogen bonds.
4. Now you have two single stranded sections BUT they have a natural tendency to re-anneal (pair up again.
5.To keep them seperate, single stranded binding proteins (SSBs) are bound.
6. Enzyme primase puts down RNA primers that will indicate to DNA polymerase III as starting point of replication.
7. DNA polymerase III (5' To 3') adds the complementary nucleotides to the 3' end of the new strand- this is called leading strand vs lagging strand (Okazaki fragments), that are built discontinuously from the replication fork.
8. DNA polymerase I excises (cuts out) the RNA primers and replaces them with appropriate deoxyribonucleotides (DNA nucleotides)
9. DNA ligase joins the gaps between the Okazaki fragments by creating phosphodiester bonds.
10. Finally, DNA polymerase I and III proof read the complementary strands.
Diagrams of the Process (Enlarge the photos in edit to see them clearly!)
Homework: Pg. 294 Q- 1, 3, 4, 6, 8 + Pg. 298 Q- 1, 3
Martha G.
November 25, 2013
Protein Synthesis
HOMEWORK ---> 7.2 Questions #1 - 6
---> If the diagrams in class is not in your possession, then copy it off of a friend!!!
Annie Vimalanathan
November 26, 2013
7.3 Translation
1) What is involved
2) Transfer RNA
Step by Step:
INITIATION:
ELONGATION:
TERMINATION:
For more information visit:
http://www.nobelprize.org/educational/medicine/dna/b/translation/translation_process.html
http://www.nature.com/scitable/topicpage/translation-dna-to-mrna-to-protein-393
http://www.youtube.com/watch?v=5bLEDd-PSTQ
Homework: Page 331 #1,2,6,8
Annie Vimalanathan
November 27, 2013
Chapter 6 quiz took place.
Homework: Bring in Field Trip Forms and Money
Saad Sheikh
November 28, 2013
Overall Process of Trancription
Control of Gene Expression
Gene expression is controlled at various levels
There are four types of control in Eukaryotic Cells
Example 1: lac Operon (negative feedback control)
Operon - a cluster of genes under the control of one promoter and operator
Operator - regulatory sequence where the repressor protein will bind
In this specific example:
Homework
pg. 339 #1-6
References
http://www.qiagen.com/products/genes%20and%20pathways/pathway%20details.aspx?pwid=452
http://www.youtube.com/watch?v=oBwtxdI1zvk
Rajavi Kanagaiyah
November 29, 2013
We had a supply in class and used the time to:
-Read page 340-345 and make notes
-Do questions 1-8 on page 345
-Work on our "build" project
Martha G.
December 02, 2013
CONTINUATION OF LESSON ON NOVEMBER 28TH!
Under Normal Conditions:
When trp is present:
HOMEWORK: Build Project !!!!
Shanukha Thivyanath
December 4, 2013
Topics Covered in Class:
- Notes on Plasmid Construction
- Notes on Plasmid Mapping
- Examples of Plasmid Mapping Questions
Plasmid Construction: How Plasmids are made
- A Plasmid contains numerous sites for restriction enzymes to act
- Are single super-coiled circular pieces of DNA inside bacteria
- Not the genomic DNA of the bacteria a specific characteristic (gives them an advantage) i.e ampicillin
-Plasmids can be designed as vehicles to transfer genetic information into a cell. These are called vectors. i.e used in insulin production
- A cell that can take up foreign DNA and express the products of the gene are called competent.
Plasmid Mapping
- A restriction map is a diagram showing the restriction enzyme sites on a plasmid
- It includes the distances (measured in base pairs) between the sites
- This allows molecular biologists to determine which plasmids are best suited to create a given recombinant DNA
To create a map. scientists first cut the plasmid with the different restriction enzymes (alone and in combination) and determine the lengths of the fragments formed by cleaving (using gel electrophoresis)
-They then use the fragment length data to create their map vectors are created by using restriction enzymes to insert the gene of interest.
Things to Consider when using Plasmids:
-Plasmids may have many restriction sites, but the one you use must occur only once (and have a sticky end if possible).
- The point of insertion should be downstream of an existing control sequence like lac or trp operons
-This allows for control of eventual gene product
- The gene of interest must have no introns or control sequences
- Genes must insert in the correct direction
Examples with Diagrams
Homework: Finish Plasmid Mapping Worksheet, front and back. **Try Level 3 Questions! Quiz is coming soon!
Saad Sheikh
December 5, 2013
Gel Electrophoresis
Homework
Reminder
Helpful Links
http://learn.genetics.utah.edu/content/labs/gel/
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
Saad Sheikh
December 6, 2013
Polymerase Chain Reaction
Purpose: to amplify a gene fragment (like one for insulin)
Applications of PCR:Restriction Fragment Length Polymorphism (RFLP)
VNTRs (variable number of tandem repeats) aka microsatelites
DNA Sequencing
Homework
page 385, 1-7
Build project
Plasmid mapping worksheets
Helpful Links
http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml
December 9,2013
Annie VimalanathanPlasmid Mapping Quiz took place
Transformation
This is the introduction of DNA from another source into a bacterial cell
The Process
1. Bacteria are put into a solution of Calcium Chloride
- This creates pores in the membrane and the calcium ions "neutralize" the negative charges on both the cell membrane and the plasmid
2. The solution is set at 0 degrees- This makes the cell membrane less fluid
3. Now foreign DNA is introduced- This DNA is also neutralized by Calcium ions
4. The solution is subjected to a sudden heat treatment of 42 degrees for about 90 seconds- This creates a draft that sweeps in the DNA into the bacterial cell
5. A nutrient broth is added and the solution incubated at 37 degrees- This helps the bacteria recover and begin to grow
6.The solution is placed into agar that contains an antibiotic.December 10, 2013
Irteza JunaidIt was an ISP today.
December 13th 2013
Tahrik RodriquesToday we performed the pGLO Lab, in hopes that our plasmid colonies glow green under UV light.
Homework: Finish the DNA "build' project that is due on Monday
Have a great weekend, and good job to all those that performed or helped in the musical!